Diisopropyl adipate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Repeated dose oral toxicity study was conducted to determine the toxic nature of the test chemical upon repeated oral administration for 28 days

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Diisopropyl adipate

EC Number:

230-072-0

EC Name:

Diisopropyl adipate

Cas Number:

6938-94-9

Molecular formula:

C12H22O4

IUPAC Name:

1,6-bis(propan-2-yl) hexanedioate

Constituent 2

Reference substance name:

Di isopropyl adipate

IUPAC Name:

Di isopropyl adipate

Details on test material:

-Name of the test material : Diisopropyl adipate
- Molecular formula (if other than submission substance): C12H22O4
- Molecular weight (if other than submission substance): 230.30 g/mol
- Substance type: Organic
-Physical state : liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were in-bred at sa-FORD Animal facility.
- Age at study initiation: 6-7 weeks at the start of treatment
- Weight at study initiation: Males: 159-191 g and Females: 139-156 g
- Fasting period before study:
- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly.
Sterilized corn cob produced from pure corn, dried and free from dust, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean. Bedding material of batch no. SPAR_27/2014 (Sparconn Life Sciences Bangalore) was incorporated.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet of batch no. 400009 and 400010 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period: 5 days prior to test item administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 23.10 °C
- Humidity (%): 49.9 to 69.40%
- Air changes (per hr): 12 times per hour and filtered adequately
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: To: September 11, 2014- November 17, 2014

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

NO data

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle to achieve desired concentration of test item. The frequency of dose preparation was daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity.

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 1000 mg/Kg bw
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required):lot nos.: MKBQ9948V and MKBG9426V
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The active ingredient (a.i.) concentration of the test item in dose formulation were analysed by validated HPLC method on day 1 and 21. Formulation was analysed for homogeneity using two samples of each lower, middle and upper layer and found homogeneous. The mean active ingredient content at 100 mg/ml concentration of Di isopropyl adipate (CAS No. - 6938-94-9) was 97.15 and 93.52 mg/ml on day 1 and day 21, respectively.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 or 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Total: 40
Main study group:
0 mg/Kg bw: 5/sex/dose
1000 mg/Kg bw: 5/sex/dose

Recovery group:
0 mg/Kg bw: 5/sex/dose
1000 mg/Kg bw: 5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The limit dose level, 1000 mg/kg body weight was selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Random
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
The observation period was 28 days. Animals in a recovery group scheduled for follow-up observations were kept for at least 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

General clinical observations of rats from the all groups were made once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter. The detailed clinical examinations were made outside the home cage, at approximately the same times, on each occasion


BODY WEIGHT: Yes
Animals were weighed during randomization, at start of treatment and thereafter weekly, till the end of experimental period. Body weight of the animals taken on the day of necropsy were used only to calculate the relative organ weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Feed consumption of all animals was determined weekly, during the treatment period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examination was conducted prior to exposure (During Acclimatization Period) to the test item and at the end of the dosing (main groups) and recovery periods (recovery group). Before ophthalmologic examination, mydriasis was induced using 1% tropicamide.

HAEMATOLOGY: Yes
Blood smears were prepared from the collected blood, stained with Leishman’s stain and were observed for Differential Leukocyte Count (%) and blood cell morphology.Blood smears for reticulocyte count (%) were stained by Brilliant Cresyl Blue Staining method and will be observed microscopically. Reticulocyte count were performed by observing the approximate 1000 RBCs with corresponding number of reticulocytes in different microscopic fields (under oil immersion) and were reported as percentage of the total number of RBCs observed.

CLINICAL CHEMISTRY: Yes
Blood collection of all surviving rats from the main groups and recovery groups was performed on the termination day, just prior to necropsy.
Animals were fasted overnight (approximately 16-18 hr) prior to blood collection. Blood was collected from retro orbital plexuses in vials without anticoagulant for clinical biochemistry, 4.0% Sodium EDTA for hematology and Sodium Citrate with 3.8% for coagulation factors. Clinical pathological evaluations of blood and serum was done as soon as possible, after collection.

URINALYSIS: Yes
At the last week of the treatment and recovery periods, all surviving rats of the main groups and recovery groups were housed in metabolic cages overnight, (Filtered water was provided ad libitum) and urine samples were collected. Color, appearance and volume of the urine was recorded after visual observation. The parameters including Blood/ blood cell, bilirubin, urobilinogen, ketone, protein, nitrite, glucose, pH, specific gravity leucocytes and microscopical parameters were analyzed using auto analyzer

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for rats from main groups, during the last week of treatment and that of recovery groups, in the last week of recovery period.

OTHER: Before necropsy, the oestrus cycle stage of all females was determined by taking vaginal smears.

Sacrifice and pathology:

All surviving animals of main and recovery groups were subjected to necropsy and detailed gross pathology evaluation. Animals were fasted overnight (approximately 16-18 hr) before necropsy. Animals were weighed, sacrificed by using over dose of CO2 and examined externally. All orifices and the cranial, thoracic and visceral cavities were opened and examined macroscopically

GROSS PATHOLOGY: Yes, Organs were kept in normal saline till they are weighed. Organs were weighed as soon as possible, after they are collected. Animals moribund sacrificed and found dead during the study, organs from these animals were weighed. These included adrenals, brain (cerebrum,cerebellum,mid brain), Epididymides, Heart, Kidneys, Liver, Ovary with oviduct, Prostate and Seminal vesicle with coagulating glands, Spleen, Testes, Thymus and Uterus with Cervix.

HISTOPATHOLOGY: Yes, Full histopathology was carried out on the preserved organs and tissues of all animals in the control and limit dose group. No organ was identified as a target organ, hence no histopathology was performed in recovery group of animals. Histopathological organs included:

Adrenals
Pancreas
Aorta Peyer's Patches
Bone (femur) with joint
Pituitary
Brain (cerebrum,cerebellum,mid brain)
Prostate and Seminal vesicle with coagulating glands as a whole
Cecum Rectum
Colon Salivary glands
Duodenum Sciatic Nerve
Epididymides
Skeletal muscle
Eyes with optic nerve Skin
Spinal Cord (cervical, mid-thoracic and lumbar)
Heart
Spleen
Ileum
Sternum with marrow
Jejunum
Stomach
Kidneys
Testes
Liver
Thymus
Lung
Thyroid with Parathyroids
Mammary glands
Trachea
Mesenteric and Mandibular lymph node
Urinary Bladder
Oesophagus
Uterus with Cervix
Ovary with oviduct
Vagina

Other examinations:

No data

Statistics:

Raw data was processed using statistical software Sigma Plot 11.0. The mean and standard deviation were calculated using the MS Excel or Sigma Plot 11.0 software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, haematology, clinical chemistry, absolute and relative organ weights) was analysed using t-test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in kidney weight

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Clinical Observations: No clinical signs were noted at 0 and 1000 mg/kg bw
Mortality: No mortality and morbidity was observed during this study

BODY WEIGHT AND WEIGHT GAIN At 1000 mg/kg bw, no effects on body weight and body weight gain were noted during conduct of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) Feed consumption of limit dose animals was unaffected during conduct of the study, as compared to control group

FOOD EFFICIENCY No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) No data available

OPHTHALMOSCOPIC EXAMINATION No data available

HAEMATOLOGY Statistically significant increase in Haemoglobin count and Mean Corpuscular Haemoglobin Concentration (MCHC) were observed at the end of treatment period, as compared to control and were not dose related. Increased Neutrophil and decreased Lymphocyte count were noticed at the end of recovery period. These changes were not test item related and may due to the pre-analytical and analytical variables or incidental

CLINICAL CHEMISTRY Changes observed in Calcium, Albumin, Bile acids, Gamma-Glutamyl Transpeptidase (GGT), Triglyceride, AST and Creatinin Kinase were not dose independent

URINALYSIS Urine parameters in test groups of both the sexes were comparable with the control

NEUROBEHAVIOUR The sensory reactivity measurements recorded during this study are commonly observed in the test system and were not attributed to the test item. There was no test item related foot splay findings. Grip strength measurement of forelimbs of the main group female animals of 1000 mg/kg bw was observed statistically significant increase in forelimb grip strength of the main group female animals at 1000 mg/kg bw was observed, as compared to vehicle control. Forelimb grip strength was comparable in the recovery group of animals. No change in hind limb grip strength was noted in main and recovery group of animals. Hence, the changes observed in main group female fore limb grip strength might be incidental and not due to test item treatment. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes throughout the experimental period; except significant increased horizontal and ambulatory counts in treatment animals, as compared to control group in males. Horizontal and ambulatory counts were comparable in the recovery group of animals. Hence, the changes observed in main group male horizontal and ambulatory counts might be incidental and not due to test item treatment.

ORGAN WEIGHTS Absolute and relative organ weights were comparable in limit dose group, compared to control group. Except, increased absolute & relative weight of kidneys in males, absolute kidneys weight in females and relative organ weight of ovaries when compared to control animals. Observed changes in kidneys weight has been linked to increased functional demand such as elevation of the glomerular filtration rate and altered tubular fluid flow and not test item related adverse effect as it was below 10 % increase compared to the concurrent control.

GROSS PATHOLOGY
External Findings: Gross necropsy of all surviving male animals from all groups showed no external abnormal changes in gross observations.

Internal Findings: Gross necropsy of all surviving male and female animals from all groups showed no internal abnormal changes. Except, animal number 5 male of vehicle control group, which showed red discoloration of all lobes of lung, which could be considered as incidental change

HISTOPATHOLOGY: NON-NEOPLASTIC Tubular degeberation of kidneys and MNC infiltration in the lungs and trachea. These observed changes are inflammatory changes and are the most common observed changes in rodents. Observed tubular hypetrophy in kidney has been linked to increased functional demand such as elevation of the glomerular filtration rate and altered tubular fluid flow and not an adverse effect related to the test item. Microscopic evaluation of uterus was comparable with oestrous cycle evaluation by vaginal smear method in the main group animals

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects were noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mortality and Morbidity

1.1      Male

Group	Treatment	Dose (mg/kg bw)	No. of Animals	Day	Observation
G1	Vehicle control	0	5	1-29	No mortality/ morbidity
G2	Limit dose	1000	5	1-29	No mortality/ morbidity
G1-R	Vehicle control -Recovery	0	5	1-43	No mortality/ morbidity
G2-R	Limit dose - Recovery	1000	5	1-43	No mortality/ morbidity

 

1.2      Female

Group	Treatment	Dose (mg/kg bw)	No. Of Animals	Day	Observation
G1	Vehicle control	0	5	1-29	No mortality/ morbidity
G2	Limit dose	1000	5	1-29	No mortality/ morbidity
G1-R	Vehicle control -Recovery	0	5	1-43	No mortality/ morbidity
G2-R	Limit dose - Recovery	1000	5	1-43	No mortality/ morbidity

 

 

 

Table 2 Clinical Signs/ Symptoms

2.1      Male   

Group	Treatment	Dose (mg/kg bw)	No. Of Animals	Day	Clincal Sign	Incidences
G1	Vehicle control	0	5	1-29	Normal	5/5
G2	Limit dose	1000	5	1-29	Normal	5/5
G1-R	Vehicle control -Recovery	0	5	1-43	Normal	5/5
G2-R	Limit dose - Recovery	1000	5	1-43	Normal	5/5

2.2      Female

Group	Treatment	Dose (mg/kg bw)	No. of Animals	Day	Clincal Sign	Incidences
G1	Vehicle control	0	5	1-29	Normal	5/5
G2	Limit dose	1000	5	1-29	Normal	5/5
G1-R	Vehicle control -Recovery	0	5	1-43	Normal	5/5
G2-R	Limit dose - Recovery	1000	5	1-43	Normal	5/5

Applicant's summary and conclusion

Conclusions:

Treatment of Wistar rats for 28 days with oral dose of 1000 mg/kg bw/day of the test chemical produced no toxicity. Findings at limit dose level of 1000 mg/kg bw/day were limited to adaptive changes in the kidneys. Hence, the No Observed Adverse Effect Level (NOAEL) of the test chemical is considered to be 1000 mg/kg/day in Wistar rats

Executive summary:

28 days repeated dose oral toxicity study was conducted to evaluate the toxic nature of the test chemical. The study was performed using male and female Wistar rats. The animals were randomly allocated to the four groups (5/Sex/Main Group and 5/Sex/Recovery Group) and were dosed daily with a limit dose of 0 and 1000 mg/Kg bw. The test material was dissolved in corn oil to obtain the appropriate dose concentration. The animals were observed for clinical signs, mortality. Ophthalmoscopic, hematological, neurobehavioral and urinalysis was also performed in addition to gross pathological and histopathological changes. Treatment of Wistar rats for 28 days with oral dose of 1000 mg/kg bw of the test chemical produced no toxicity. Findings at limit dose level of 1000 mg/kg bw were limited to adaptive changes in the kidneys. Hence, the No Observed Adverse Effect Level (NOAEL) of the test chemical is considered to be 1000 mg/kg/day in Wistar rats.